The present application is the national stage under 35 U.S.C. 371 of PCT/IT98/00202, filed Jul. 17, 1998.
The present invention has as its object new compounds usable in the therapy of a series of human pathologies such as tumoral growth and metastatization, atherosclerosis, multiple sclerosis, Alzheimer""s disease, osteoporosis, rheumatoid arthritis and other inflammatory diseases. Said compounds in fact, following in vitro experiments extensively described in the following chapters, showed a remarkable inhibitory capability on certain human enzymes, the zinc-dependent metalloproteinases, which have been related with such pathologies (see for example: xe2x80x9cInhibition of matrix metalloproteinases. Therapeutic potentialxe2x80x9d - Annuals N.Y. Acad.Sci. 732 (1994)). Thus although an integration of experimental data with adequate evidence in vitro is naturally necessary, the results collected already allow to expect their usability in specific therapies. Moreover such inhibitory capacity was originally demonstrated also in a series of zinc-dependent metalloproteinases extracted from snake venoms, also denominated xe2x80x9chemorrhaginesxe2x80x9d for their capacity of inducing extensive internal herorrhagies in victims of snake bites, and constitute the most damaging agent in the venomous mixtures elaborated by Crotalidae and Viperidae. Thus their usability also in preparing specific antidotes against venom of Crotalidae and Viperidae seems evident.
The design and synthesis of such compounds in fact constitutes the last step in a long course of research, based on the study of the structure and action mechanism of certain particular zinc-dependent metalloproteinases called hemorhagines.
The article IL FARMACO (1993), 48 (9), 1271-7 shows that in the study of peptidic inhibitors a conformationally restricted model of compound tested on proteinase II from Crotalus Adamanteus snake venom has a sensibly lower inhibitory activity than that of related substrates. This result indicates that the structure of the inhibitor compound has direct influence on the fitting and bind at the enzyme active site and is not foreseeable a priori.
The so-called Hemorrhagic Factors of Hemorrhagines constitute a very important class of enzymes detected in the venom of snakes belonging to the Crotalidae family. They are structurally of use to the snake as they rapidly induce extended internal hemorrhagies in victims, causing circulatory collapse and preventing the victim from escaping its fate. The mechanism of the hemorrhagic action is due to the particular ease with which the enzymes are capable of degrading a large number of filiform proteins which bind the various vasal endothelid cells, allowing the elements of the blood to escape from the vessels. Although their molecular weights differ greatly, the hemorrhagines maintain however some fixed characteristics on the catalytic site, in the way that Zinc bonds with certain amino acids of the proteic chain, and in the way in which they attack the proteins of the basal membrane of blood vessels. They also seem to have in common the mechanism which ensures the protection of the snake""s organism from the toxic effects of its own metalloproteinases, which seems based on the production of tripeptides capable of functioning as competitive inhibitors, interacting with the active site of the enzyme containing Zinc (Biomed.Biochim.Acta 50, 769-773, 1991).
Now the presence of Zinc in the active site of the enzyme constitutes one of the most interesting aspects of the study of Hemorrhagines. In fact this characteristic is not exclusively theirs, but characterizes a wide number of proteolytic enzymes which perform a series of important and diversified physiological and pathological functions in other animal organisms, evolutively also quite distant from each other. In relation to this a comparative study was carried out to determine possible similarities and differences in structure.
By studying the sequences of residues of the proteic chains and the amino acids involved in Zinc bonding it has been possible to obtain a sort of xe2x80x9cfamily treexe2x80x9d for this family of proteinases (see for example FEBS Letters 312, 110-114, 1992 and Developmental Biology 180, 389-401, 1996): it has thus been seen that the active site of enzymes belonging to living beings quite distant from each other, such as Astatin (extracted from a river crustacean), Seratin (obtained from a microorganism), Matrixines (present in the organism of mammals) and those of Hemorrhage Factors of snake venom, in reality differ only in one of the four amino acids binding Zinc. Thus in spite of the fact that there are strong differences in the rest of the proteic structure, they can be considered to be in some way evolutively correlated. This is particularly interesting when considering the fact that the functions performed by these enzymes are not in any way analogous. In fact it has been ascertained that proteolytic enzymes of snake venom if on one hand are very similar and have thus allowed the definition of a proper new family of proteinases: snake venoms metalloproteinases (see for example Biol.Chem. Hoppe-Seyler 373, 381-385, 1992), on the other hand, they do not show any functional similarity with any other protein of the plant or animal world. In particular an extremely relevant fact is the difference between the functionality of hemmorhagines and those of Human Matrixines, which exercise important effects on cell migration and on the reconstruction of damaged tissues. Moreover, while Matrixines are released in the form of xe2x80x9czimogensxe2x80x9d (that is, inactive enzymes which must be made functional through other proteinases intervention), and can be inhibited by particular proteins (TIMP), hemorrhagines are immediately active at the moment of dilution in the blood flow. In spite of such structural and functional differences the Applicant has determined the existence of a close correspondence between the inhibition of snake hemorrhagines and the pharmacological results obtained on animal models in which the patogenous agent is presumed to be a zinc-dependent metalloproteinase produced by the tissues of the mammal. Such correspondence seems the consequence of a structural resemblance existing albeit only in the active site between two different types of metalloproteinases and, based on this, the Applicant has developed a method for the selection of compounds for potential therapeutic use in man (Italian patent application RM95A000557; European patent application EP0758021).
Many mammal zinc-dependent metalloproteinases in fact have been related with pathological situations, some of which have been mentioned above. For example gelatinases seem involved in tumoral metastatization, while collagenases have a pathogenic role in arthritic phenomena.
EP-A-0 401 963 describes phosphonopeptides showing an inhibitory activity in regard to enzymes of the collagenase family and as such the compounds are considered useful in the treatment of arthritic and other diseases.
Certain compounds which inhibit matrixines have begun the phases of clinical development in patients suffering from tumour or arthritis: however they are usually scarcely absorbed when administered orally, and are constituted by hydroxamates, compounds which can present toxicity problems in chronic administration.
Finally, a new family of zinc-dependent metalloproteinases was recently identified, localized on the cell membrane, which possess the same proteic domains of hemorrhagines, and are thus considered their closest relatives (see Developmental Biology 180, 389-401, 1996). These proteinases, called ADAM (A Disintegrin and A Metalloproteinase Domain), are correlated with the functionality of the reproductive apparatus, but are also responsible for releasing TNF-a (Tumor Necrosis Factor alfa) and ACE in the circulation and seem correlated to SNC diseases, including Multiple Sclerosis.
The aim of the present invention is to supply compounds with pharmacological activity towards human pathologies, which have been securely related with the enzymatic activity of zinc-dependent metalloproteinases.
The strategy followed to solve this problem was to take as starting-points the ascertained resemblance in the structure of the active site existing between Hemorrhagines and other mammal zinc-dependent metalloproteinases, and the mechanism of protection of the snake against the effects of its own venom, based on the production of peptides inhibiting the enzymatic activity of Hemorrhagines themselves.
Based upon these data, the synthesis of new compounds capable of acting as inhibitors of the snake""s proteinases was designeded, supposing that, given the structural resemblance in the active site, they could also act as inhibitors of human zinc-dependent metalloproteinases.
Therefore, a first step consisted in designing these compounds on the basis of the three-dimensional characteristics of the active site of a Hemorrhagine resolved on X-ray, Adamalysin II, and of the known data concerning the xe2x80x9cnaturalxe2x80x9d inhibitors of such enzyme.
In this way in fact it is possible not only to visualize the relative structure of each single domain of the protein, but also to verify the relationship of domains within the quaternary structure of the protein. It is therefore possible to obtain some structural data which together with the enzymatic data offer a significant contribution to the comprehension of the action-inhibition mechanism of a given protein. It has thus been possible to design and build certain compounds potentially capable of binding the active site and of acting as inhibitors of the protein itself.
As an outcome of this phase it has been possible to arrive at a definition of these xe2x80x9cpeptido-mimeticxe2x80x9d compounds, that is, similar to peptides, but lacking at least one of the bonds that make molecules easily attackable by the proteolytic enzymes, with an important characteristic in the substitution of the residue of the terminal tryptophan with the analogous phosphonate.
The compounds according to the present invention can be represented according to the following general formula: 
in which R can be H, or CH2xe2x80x94C6H5, and Rxe2x80x2 can be a saturated or aromatic ring formed by five or six members, of which one at least is not carbon, but can be selected among nitrogen, oxygen and sulphur. An example is supplied by the following structures: 
Such compounds were then obtained through two different synthesis schemes, confluent into one another in the final phase.
In Diagram 1, diethylester of (1)-phosphotryptophan (1) obtained by using a modified version of the method described above (Subotkowski, J., Kowalik J., Tyka R., Mastalerz P. Pol.J.Chem. 1981, 55, 853-857; Rogers R. S., Stern M. K. Synlett. 1992, 708), (i.e., the reduction of 1-hydroxymino-2-(3-indolyl) ethane phosphonate with aluminium amalgam in presence of aqueous ammonia), is reacted with a pseudo-peptide, obtained by acylation of leucin with Rxe2x80x2xe2x80x94COOH acid, where Rxe2x80x2 represents a saturated or aromatic ring, formed by five or six members, of which at least one is not constituted by carbon, but can be selected among nitrogen, oxygen and sulphur. The resulting diethyl esters (3) are thus transformed into the corresponding free phosphonic acids which were isolated, purified and kept as cyclohexylmaine salts (4). 
In the specific case of compound 5, said compound was obtained and isolated as an inner salt. This occurs also with other saturated rings of 5 or 6 member containing nitrogen. 
The conditions under which the aforementioned experiments were carried out are indicated by symbols in the diagram and are the following: (i) DCC, 1-HBT, THF, 15 h, 53-73%; (ii) N,O-bistrimethylsilyl-acetamide, Me3SiI, CH2Cl2, 25xc2x0 C., 2 h; (iii) C6H11NH2AcOEt, 40-86%; (iv) 10% Pd/C, EtOH, 25xc2x0 C., 2 h, 85%.
In Diagram 2 instead indoleacetic acid (6) is converted into benzyl diethyl-ester (8) of phosphotryptophan. 
The compound thus obtained (8) is then reacted with the pseudo-peptides already used in the synthesis scheme described previously. 
The removal of only one of the two benzyl groups produces monobenzylester (10). 
The reaction conditions are here too summarily distinguished by symbols and are listed below: (i) ClCOxe2x80x94COCl, CH2Cl2, 0.3% DMF, refl, 30 min; (ii) Me3Si-O-P (Oxe2x80x94CH2xe2x80x94C6H5)2, CH2CL2, xe2x88x9218xc2x0 C., 1 h; (iii) NH2OH HCL, C5H5N, EtOH, from xe2x88x9218 to 4xc2x0 C., 15 h 46%; (iv) AlHg, NH4OH, EtOH, 25xc2x0 C., 2 h, 68%; (v) Rxe2x80x2xe2x80x94COxe2x80x94Leuxe2x80x94OH, ixe2x80x94Buxe2x80x94Oxe2x80x94COxe2x80x94Cl, NMM, THF, xe2x88x9220xc2x0 C., 88%; (vi) AlHg, NH4OH, EtOH, 25xc2x0 C., 4days, 58%.
The therapeutic activity of pseudo-peptides thus obtained was verified applying the method previously devised by the Applicant (Italian Patent application RM95000957) taking into consideration the specific characteristics of the case.
The compounds thus obtained were first subjected to experiments to test their actual capacity of inhibiting enzymatic activity of snake venom metalloproteinases, having been designed on the relative three-dimensional structure of such metalloproteinases, with respect to the capacity of bonding to the enzymes"" active site and thus of acting as competitive inhibitors.
This property was verified in particular by inhibition tests in vitro on metalloproteinase Adamalysin II purified from Crotalus Adamanteus venom, a protein of which the three-dimensional configuration is entirely known, which was also selected because among snake metalloproteinases it is the one closest to human metalloproteinase, due to the remarkable homology of the primary aminoacid sequence which it presents with the enzyme which releases TNF-a in man (TACE) in the active site. The results extensively described in example 5 indicate the existence of a good inhibitory capacity in all tested compounds, some of which show also a remarkable power of action (see infra table I). Because of the resemblance between Adamalysin II and TACE, these results are indicative per se also of a possible pharmacological activity of the compounds against TNF-a.
The next step consisted therefore in testing the inhibitory capacity of such compounds also in relation to human metalloproteinases. Reference proteinases were significantly selected for this reason as neutrophile Collagenases and purified Gelatinases A from human cell cultures.
Gelatinase A also know as MMP-2, is in fact an enzyme belonging to the Matrixin family which have been shown to be produced in great quantity in many pathological situations, and believed to be primarily responsible for the migration of tumoral cells from the blood towards tissues affected by metastasis phases.
Likewise also neutrophile Collagenases (denominated MMP8), also belonging to the matrixin family, has been related with a large number of pathological situations. In particular it is considered primarily responsible for the destruction of cartilage which is observed in cases of chronic inflammation. Consequently, it is quite clear that the identification of inhibitors of the activity of both these proteins must be considered a first and important step to elaborate new efficient therapies for these pathologies.
With reference to these considerations, testing has been carried out on neutrophile Collagenases and Gelatinases A themselves for assessing the effective synthesized compounds capacity to bond to the active site of human metalloproteinase, and so to act as competitive inhibitors.
The results extensively described in examples 5 and 6 have shown the existence of a good (albeit varying from compound to compound) inhibitory capacity of such compounds also for these metalloproteinases. Also considering the fact that the two proteinases perform functions which are also performed by other matrixines, the results obtained for both must therefore be considered as indicative also of the potential inhibitory capacity of the pseudo-peptides object of the present invention on other zinc-dependent matrixines implicated as pathogenetic agents, in many pathological situations in man.
These capacities are thus indicative of a potential use of pseudo-peptides as powerful therapeutic agents, whose efficacy and pharmacological usefulness is enhanced by their peculiar chemical characteristics. The modifications of the peptidic structure of these compounds are likely to make them resistent to gastric proteolytic enzymes, and therefore they can be considered suitable for oral administration. Moreover the substitution of the terminal tryptophan residue with the analogous phosphonate remarkably increases the inhibitory activity on enzymes, without introducing risks of systematic toxicity of molecules presently subjected to clinical testing in tumors and arthritis. In fact they are based on hydroxamate compounds which have great power in bonding zinc, but which after prolonged administration can introduce in the organism an accumulation of hydroxylamin, a potentially cancerogenous agent. The compounds object of the present invention instead, being phosphonates, do not present risks of dangerous side-effects, as is demonstrated by drugs of this category which are on the market now.
With reference to all the above, object of the present invention are compounds of general formula: 
in which R can be H, or CH2xe2x80x94C6H5, and Rxe2x80x2 can be a saturated or aromatic ring of five or six members, of which at least one is not carbon, and can be selected among a group including nitrogen, oxygen and sulphur. A particularly preferred case is that in which Rxe2x80x2 is selected from the group including: 
Also object of the present invention is the use of the same as inhibitors of enzymatic activity of at least one of the zinc-dependent metalloproteinases extracted from the venom of snakes belonging to the families of the Crotalidae and of the Viperidae also denominated hemorrhagines (in particular Adamalysin II), and/or at least one of the zinc-dependent metalloproteinases of human origin, of which the active site presents a three-dimensional structure analogous to that of the said snake metalloproteasis (in particular the neutrophile Collagenases, Gelatinases A and the ADAM).
Consequently to all observations reported so far the use of such compounds as pharmaceuticals for the therapeutic treatment of all human pathologies in which the pathogenic mechanism or in which the symptomatology has been demonstrated to include at least one zinc-dependent metalloproteinase, and relative pharmaceutical compounds containing them must also be considered. In particular, reference is made to tumoral growth and metastasization, atherosclerosis, multiple sclerosis, Alzheimer""s disease, osteoporosis, hypertension, rheumatoid arthritis, and other inflammatory diseases.
A further object of the present invention is the process for producing the (1)-phosphotryptophan diester as the starting product for the synthesis of the compounds previously described, including as an essential operation the reduction of 1-hydroxymino-2-(3-indolyl) ethane phosphonate by adding an amalgam of aluminum in presence of aqueous ammonia.
A general description of the present invention has been made so far. With the aid of the following example, a more detailed description of specific embodiments will now be given, in order to give a better understanding of the objects, characteristics, advantages and operating methods of the invention. Such examples serve merely to illustrate and do not limit the scope of the present invention.